A significant number of therapeutic agents for the treatment of ischemic heart disease target G protein coupled receptors (GPCR) and protein kinases. Up to four subtypes of one GPCR, the adenosine receptor, may be expressed in mammalian myocardium and all four subtypes have been shown to modulate protein kinase signaling. Published and preliminary findings from the applicant's laboratory indicate that adenosine A2a receptors modulate the cardioprotective effects of adenosine A1 receptor stimulation. Additional preliminary findings indicate that interactions between the adenosine A1 receptor and the 4-opioid receptor modulate the cardioprotective effects of each receptor. The cardioprotective effects of both adenosine A1 and 4-opioid receptors are dependent on multiple protein kinases. The overall hypothesis of this proposal is that the interactions between adenosine receptor subtypes and between adenosine and opioid receptors in mediating their cardioprotective effects are due to the modulation of subcellular protein kinase signaling. Specific Aim 1 will determine the role of interactions among adenosine receptor subtypes and opioid receptors in reduction of myocardial ischemia-reperfusion injury in intact hearts and isolated myocytes. Specific Aim 2 will determine whether the cardioprotective effects of adenosine receptor subtype and opioid receptor interactions are due to modulation of subcellular protein kinase signaling in intact myocardium and isolated cardiomyocytes. Specific Aim 3 will delineate the effects of interactions among adenosine receptor subtypes and opioid receptors on the modulation of myocyte contractility, intracellular calcium handling and mitochondrial function in isolated cardiomyocytes. Studies will be conducted in wild-type mice and mice with deletions of adenosine A1 receptors, A2a receptors, 4-opioid, and :-opioid receptors. Receptor signaling via the PKC, MAPK and AKT pathways will be examined in nuclear, cytosolic, mitochondrial and membrane subcellular fractions generated from normal and ischemic-reperfused isolated mouse hearts and isolated ventricular myocytes. Receptor- induced modulation of subcellular protein kinase signaling in intact hearts will be correlated with myocardial infarct size and ventricular function. Subcellular signaling in isolated myocytes will be correlated with myocyte contractility, intracellular calcium homeostasis and mitochondrial function. Although protection of ischemic myocardium appears to be mediated via activation of multiple protein kinases, conditions such as myocardial hypertrophy, heart failure, and ventricular remodeling following open heart surgery are also associated with increased protein kinase activity. The results of these in-depth studies of GPCRs and their interactions on subcellular protein kinase signaling in normal and ischemic myocardium may facilitate the development of new therapies for the treatment of the ischemic heart.